Discovery of improved therapies for breast cancer will require assay systems that accurately recapitulate the cellular signaling of intact mammary tissue. To reconstitute mammary tissue structure and function faithfully in vitro, we will develop a microfluidic device for co-culturing human mammary epithelial and stromal cells in adjacent, separately addressable compartments that can be directly observed by microscopy. This assay system will enable identification and validation of molecular drug targets in a controlled microenvironment specifically designed to mimic the in vivo tissue context of mammary tumorigenesis. Three dimensional organotypic and heterotypic culture methods using cells imbedded in gel matrices have been developed to model ECM effects and paracrine signaling in the breast, but their reliance on traditional cell culture systems (e.g. tissue culture dishes) limits their accuracy and utility. In Phase I, we will fabricate a microfluidic device designed specifically for reconstructing and monitoring mammary tissue structure and function. Existing methods for 3D culture of epithelial organoids in proximity to stromal cells will be optimized for the device to produce a Microfluidic Reconstituted Mammary Tissue System (uF-rMTS). The scale and fluid control properties of the uF-rMTS will provide a more native microenvironment than the bulk aqueous environment of current tissue culture systems and allow better replication of in vivo signaling phenomena. In Phase II, the microfluidics device will be multiplexed to increase throughput, additional normal and malignant cell lines will be incorporated, and methods will be developed to probe paracrine signaling by growth factors and steroid hormones. Development of a system for probing tumorigenic signaling processes in the context of intact mammary tissue will accelerate the discovery of more effective therapies for breast cancer.